The early simian virus 40 (SV40) gene product, large tumor (T) antigen, is responsible for the initiation of viral DNA replications and the autoregulation of early gene expression through direct protein-DNA interactions. We investigated the role of T-antigen in late viral gene expression, independent of its function in amplifying templates through DNA replication. SV40 DNA was transfectd into BSC-1 and COS-1 cells and in the presence of inhibitors of DNA replication. Electrophoretic immunoblot analysis indicated that both the onset and the extent of SV40 late gene expression is increased in COS-1 cells, which constitutively express SV40 T-antigen. Blot hybridization analysis of poly(A)-selected RNA demonstrated that the level of synthesis of the major late structural protein, VP-1, in COS-1 cells was due to increased transcription. Similar results were obtained when plasmids that contain the SV40 late gene but lack both the origin for viral DNA replication and the early gene coding region were transfected onto COS-1 cells. Using lines of SV40-transformed monkey kidney cells that express altered T-antigen, we found that enhanced expression of the late gene product is correlated with the ability of T-antigen to bind SV40 DNA. Cotransfection in CV-1 and HeLa cells of recombinant DNA plasmid containing the SV40 large T-antigen and a plasmid containing the SV40 late gene region induces transcription from the SV40 late promoter. These results indicate that large T-antigen plays a role in the stimulation of late viral gene expression. In vivo promoter competition studies suggest two important aspects of T-antigen-induced SV40 late transcription. First, the transcriptional activation requires direct binding of T-antigen to sites I and II. Sedond, T-antigen activation of SV40 late transcription may require removal of negative transcriptional control factors from the SV40 late promoter.